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The use of cationic liposomes to deliver nucleic acids has shown great promise; however, their therapeutic potential is greatly limited by their in vivo instability. Upon systemic administration, cationic liposomes are prone to plasma protein adsorption, leading to RES recognition and rapid clearance from blood circulation. Surface modification using hydrophilic polymers, usually PEG, is known to prolong the circulation half-lives of liposomes in vivo. PEG is required for increased liposome stability, but it is undesirable once at a target tissue since its presence significantly inhibits the release of the encapsulated agent. The development of cleavable PEG that can be removed in response to a specific trigger once at the target site may be one strategy to overcome this problem. Here, we report the development of PEGylated peptide-lipid conjugates sensitive to the proteolytic enzymes found in tumour cells, HLE and MMP-2. The synthesis was carried out by conjugating an enzyme substrate peptide between PEG and a lipid, enabling a series of enzyme-sensitive PEGylated peptide-lipid conjugates to be used for liposomal nucleic acid delivery. Although enzymatic degradation of the peptide linker resulting in the detachment of PEG was not observed with the analytical techniques used, the in vitro result showed enhanced pDNA transfection efficiency by the nanoparticles containing the PEGylated peptide-lipid conjugates in response to the enzymes, compared to the controls. Physicochemical characterisation showed the nanoparticles have small diameters and high nucleic acid encapsulation efficiency. The nanoparticles with lower zeta potential were found to exhibit enhanced stability in serum and minimum toxicity. Further investigation in a siRNA system revealed the ability of the PEGylated peptidelipid nanoparticles to significantly knockdown the target protein in response to enzymatic activation. Thus, the results from these studies demonstrated the possibility of using the enzyme-sensitive PEGylated peptide-lipid conjugates to improve nucleic acid delivery.